Many peptides, polypeptides, and proteins (collectively, “target peptide(s)”) can be produced via recombinant means. Recombinant protein production has been established in a variety of expression systems. Such expression systems, include strains of bacteria and fungi as well as mammalian and baculovirus or insect cells. These expression systems are not without technical problems. One problem is the recovery or separation of the target peptide from the system as a whole.
Isolating a target peptide from native or host cell/expression system proteins and other cellular products is a significant hurdle in expression system utility. Consider, for example, yeast systems employed for synthesis of target peptides such as human growth hormone, interferons and the like. The biological activity (and potential utility) of the target peptide is dependent upon the target peptide's assumption of specific secondary and tertiary structural conformations. In many instances, the secondary and tertiary structural conformation sought is that duplicative of a the native state configuration.
In some expression systems, target peptide accumulate within the host cells as insoluble aggregates. Recombinant proteins expressed are known to accumulate in cytoplasm as insoluble aggregates known as inclusion bodies. (F. A. O. Marston, Biochem. J. 240:1-12 (1986); C. H. Schein, Biotechnology 7:1141-1149 (1989)). This is particular noted in bacteria and yeast expression systems. The effectiveness of an expression system turns, in part, on recovery of Target peptide in a soluble active form with particular reference to native state configuration
Peptides, polypeptide, and proteins are chains of amino acids linked by peptide bonds. As a general biological principal, the behavior of a peptides, polypeptide, or proteins in a chemical or biological system is effected by or related to its (i) amino acid composition, (ii) configuration (i.e., the three dimensional arrangement of amino acid side groups in a particular order) and (iii) conformation (i.e., the three dimensional arrangement of side groups in amino acids which can freely rotate into different positions without breaking bonds). In a given biological system a peptides, polypeptide, or protein of that system is folded into a specific three dimensional structure. Without being bound by ant particular theory, it is believed that a particular three dimensional structure is determined by the thermodynamic forces, stearic considerations, covalent disulfide bonds, if any, and noncovalent interatomic forces (i.e., charge, hydrogen bonding and hydrophobic interactions).
In the isolation of target peptide from recombinant expression systems, preservation of bioactivity and or native state configuration has been a problem in prior art methods. A target peptide that is recovered in a non-native state configuration is potentially of altered bioactivity. Altered bioactivity is variously presented as more active in some reactions and less active in others. In some instances, a longer half-life will enhance the total activity of a target peptide even if the instantaneous activity is less than a naturally occurring peptide. A number of theories have been advanced to explain target peptide resulting from expression systems in non-native state configuration. One view is that the environment of the expression system does not provide conditions for proper “folding” of the target peptide. Reports in the art suggest that the tertiary structure of peptides and proteins is a direct result of the sequence, (secondary structure). Under some conditions, peptides and proteins in an inactive configuration of configuration of reduced bioactivity configuration are induced to adopt (more) bioactive or native state configurations.
Again, without being bound by any particular theory, it is thought that some biologically inactive peptides, polypeptides or proteins are inactive due to being “frozen” in a particular conformation as a result of “extraneous” or “incorrect” cystine disulfide bonds. In some instances “incorrect” cystine disulfide bonds arise during target peptides expression in a given expression system. By this theory, as the number of cysteine residues in Target peptides increases, the probability that disulfide bonds will properly form decreases. A disulfide bond is a covalent cross-link between two cysteine residues that have been oxidized to form cystine. Disulfide bonds are cleaved by reducing agents [e.g., DTT or beta-mercaptoethanol] to form sulfhydryl or thiol groups which are rather unstable. Disulfide bonds are largely permanent in the absence of unusual chemical manipulation. A denaturation/renaturation step is unlikely to restore bioactivity when the basis of inactivity is non-native state disulfide bonds. Disulfide bonds largely exclude further conformational changes and thus exclude adoption of native state configuration (or some other desirable tertiary configuration).
Reported difficulties associated with recovery of biologically active polypeptides containing multiple disulfide bonds have been so severe that polypeptide analogs of significant proteins have been “designed” for expression on the basis of their greater potential for recovery in a bioactive state absent incorrect disulfide bonds rather than for enhanced or prolonged therapeutic activity. As one example, the general inability to recover troponin subunit polypeptides in biologically active form prompted construction of genes for expression of various troponin analogs wherein undesired disulfide bond formation was precluded by replacing cysteines with other amino acids. Fujita-Becker et al., “Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli.,” J. Biochem. 114:438-44 (1993). For polypeptides with two or more cysteine bonds, however, such techniques will be of limited effect.
Note is made of the following publications:    1. Stryer, Biochemistry, 2d Ed., 32-36 (1981).    2. U.S. Pat. No. 5,340,926, Lowe et al. “Process for the recovery of recombinantly produced protein from insoluble aggregate.”    3. U.S. Pat. No. 4,511,502, Builder et al. “Purification and activity assurance of precipitated heterologous proteins”    4. U.S. Pat. No. 4,511,503, Olson et al., “Purification and activity assurance of precipitated heterologous proteins.”    5. De Bernardez, “Refolding of recombinant proteins.” Curr. Opin. Biotechnol. 9:157-163, (1998)    6. Fischer, “Renaturation of recombinant proteins produced as inclusion bodies.” Biotech. Adv. 12:89-101 (1994).    7. Guiseet al., “Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies.” Mol. Biotechnol. 6:53-64 (1996)    8. Hlodan et al., “Protein folding and its implications for the production of recombinant proteins.” Biotechnol. Genet. Eng. Rev. 9:47-88 (1991)    9. Jaenicke R, et al. “Refolding and association of oligomeric proteins.” Meth. Enzymol. 131:218-50 (1986)    10. Marston, “The purification of eukaryotic polypeptides synthesized in Escherichia coli.” Biochem. J. 240:1-12 (1986).
Transgenic plants have proven to be a versatile expression system, successfully used for antibody fragments, IgG and secretory IgA antibodies. Plants are higher eukaryotic organisms with an endomembrane system. Plants fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells. Notably, plant systems glycosylate proteins.    11. Sanchez_Navarro et al., “Engineering of alfalfa mosaic virus RNA 3 into an expression vector,” Arch Virol. 146(5):923-39 (2001).    12. Kusnadi et al., “Production and purification of two recombinant proteins from transgenic corn.” Biotechnol Prog 14(1):149-55 (1998)    13. Streatfield et al., “Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants,” Trends Plant Sci 6(5):219-26 (2001).
Eggs systems, conveniently chicken eggs systems, produce recombinant protein with particular reference to human therapeutics such as antibodies.    14. Mohammed et al., “Deposition of genetically engineered human antibodies into the egg yolk of hens,” Immunotechnology (1998) 4(2):115-25.    15. Zajchowski, et al., “Incorporation of genetically modified cells in chicken chimeras,” Methods Mol Biol 36:391-7 (2000).Also    16. Suttnar et al., “Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.” J. Chromatogr. B. Biomed. Appl. 656:123-6 (1994).
In the isolation of target peptides from a given expression system, protein solubilization from inclusion bodies is a significant concern. In some systems, protein aggregates are solubilized with chaotropic reagents such as guanidine hydrochloride and urea; with thiol compounds such as beta-mercaptoethanol and dithiothreitol; with inorganic salts such as potasium or sodium thiocyanate, lithium bromide and sodium iodide; organic solvents; formamide, dimethylformamide, dichloro- and trichloroacetic acids and their salts; powerful detergents such as sodium dodecyl sulphate and cetyltrimethylammonium chloride; increasing temperature, strong alkalis with salts or a combination of chaotropic reagent and strong alkali solutions; and high pressure and ultrasonic homogenization also denature protein molecules.
All these chemical compounds and physical forces cause dissociation of S—S bonds, which are essential for maintaining the conformation and rigidity of active sites, and biological activity. Furthermore, strong alkalis cause hydrolysis of peptide bond or amides, hydrolysis of arginine, loss of amino acids by alpha- and beta-elimination and racemization, and formation of double bonds or modified amino acids. Salts such as sodium chloride, sodium acetate and sodium sulfate compete with the proteins and stabilizers for the water molecules and their large positive change in chemical potential destabilizes the system causing protein precipitation rather than solubilization. It has been reported that 6M Guanidine Hydrochloride and 8 M Urea are commonly used to cause such S—S bond or disulfide bridge dissociation. Dissociation of these essential S—S bonds leads to loss of biological activity of some proteins. Thiol compounds such as Beta-mercaptoethanol and Dithiotreitol (DTT) cleave disulfide bonds by reduction of S—S bonds to the —SH form of cysteine residues in the denatured protein. Such compounds are usually added to solutions of chaotropic reagent during denaturation. Furthermore, in methods constituting the prior art, to refold the recombinant polypeptide into a biologically active product, the denaturant must be removed from the denatured protein, a slow, complex and difficult process, which usually results in protein precipitation and low yields. It is also required that SH groups are re-oxidized during refolding to produce a biologically active polypeptide. As reported, this is achieved using Cysteine and Cysteamine, or Glutathione in its oxidized and reduced form to provide the appropriate redox potential allowing the formation and reshuffling of disulfides. The removal of the denaturant by dialysis or direct dilution often results in protein re-aggregation rather than fold resulting in accumulation of inactive species and further complicating the purification process. To slow down the aggregation process refolding is usually performed at very low protein concentrations, in a range of 10-100 ug per ml. In addition, only small quantities of this material contain biological activity. Consequently the solubilization and refolding processes have been the main problem in the production of high quantities of recombinant polypeptides and the many methods described cannot be applied to any polypeptide as general methods. In summary, the solubilization of inclusion bodies with strong chaotropic reagents and/or strong alkalis, detergents, salts and/or high temperatures as well as the removal of denaturants and the subsequent protein dilution in the presence or absence of thiol compounds to induce refolding of the protein into a biologically active form, have been the rule for recovery of recombinant proteins that have been over-expressed in microbial hosts.